Total phenolic content - Folin-Ciocalteu reducing capacity (FCR assay) Antiradical activity against DPPH radical Ryszard Amarowicz Institute of Animal Reproduction and Food Research
General chemistry of the F-C method Na 2 WO 4 /Na 2 MnO 4 (phenol - MoW 11 O 40 ) -4 Mo(VI) (yellow) + e - Mo(V) (blue) Folin O., Ciocalteu V.: Tyrosine and tryptophan determination on proteins. J. Biol. Chem., 1927, 73, 627-649.
Modyfication of the F-C method by Singleton & Rosi Molybdotungstophosphoric hereopolyanion reagent: 3H 2 O P 2 O 5 13WO 3 5 MoO 3 10H 2 O and 3H 2 O P 2 O 5 14WO 3 4 MoO 3 10H 2 O Singleton V.L., Rosi J.A.: Colorimetri of total phenolics with phosphomolybdicphosphotungstic acid reagent. Am. J. Enol. Vitic., 1965, 16, 144-158.
Absorption spectra produced by wine (W) and gallic acid (G) with Folin-Ciocalteu (C) and Folin-Denis (D) reagent Singleton V.L., Orthofer R., Lamuela-Raventos R.M.: Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol., 1999, 299, 152-178.
The explicit conditions of the Singleton-Rossi method are as follows: Mix 1 ml of sample (properly diluted) with at least 60 ml of water and 5 ml of F-C reagent; after 30 s and before 8 min, add 15 ml of Na 2 Co 3 ; mix and bring to 100 ml total volume with water; incubate for 2 h at 75 o F and measure absorbance.
The F-C method suffers from a number of interfering substances: Sugars Aromaric amines Sulfur dioxide Ascorbic acid Organic acid Fe(II)
Nonphenolic organic substances that react with F-C- reagent includd: Addenine Adenosine Alanine Anilin Aminobenzoic acid Ascorbic acid Benzaldehyde Creatinine Cysteine Cytosin Dimethylaniline Diphenylamine EDTA Fructose Guanine Guanosine Glycine Histamine Histidine Indole Proteins Pyridoxine Sucrose Sulfanilic acid Thiourea Thymine Thymidine Trimethylamine Tryptophan Uracil Uric acid Xanthine
Some inorganic substances may also react with F-C reagent: Hydrazine Hydroxyammonium chloride Iron ammonium sulfate Manganese sulfate Potassium nitrite Sodium cyanide Sodium metabissulfite Sodium phosphate Sodium sulfite Tin chloride
Molar absorptivity in F-C assay from selected phenols and potentialy interfering substances Compound Molar absorbance (1000 ) Molar absortivity per reactive group Compound Molar absorbance ( ) Molar absortivity per reactive group Phenol p-coumaric acid Tyrosine Catechol Chlorogenic acid DOPA Ferulic acid Vanillin Pyrogallol Gallic acid Sinapic acid Phloroglucinol p-hydroquinone Resorcinol (+)-Catechin Kaempferol 12.7 15.6 15.7 22.5 28.9 24.6 19.2 14.9 24.8 25.0 33.3 13.3 12.8 19.8 34.3 29.6 12.7 15.6 15.7 11.2 14.4 12.3 19.2 14.9 12.4 12.5 16.6 13.3 12.8 19.8 11.5 14.8 Quercetin Quercitrin Malvin o-aminophenol p- Aminophenol p- Methylaniline Flavone Flavanone 3-Hydroxyflavone 4-hydroxycoumarin Acetylsalicylic acid D-Fructose Ascorbic acid Ferrous sulfate Sodium sulfite 48.3 44.8 40.5 31.1 21.8 11.6 0.1 1.9 3.5 0.1 0.2 0.0 17.5 3.4 17.1 16.1 14.9 20.2 15.6 10.9 11.6 0.1 1.9 3.5 0.1 0.2 0.0 17.5 3.4 17.1 Singleton V.L., Orthofer R., Lamuela-Raventos R.M.: Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol., 1999, 299, 152-178.
Callibration curves for F-C method Compound Regression r Range Compound Regression r Range sinapic acid y = 2.6715x + 0.0101 0.9996 0.0-0.3 chlorogenic y = 3.2033x + 0.0352 0.9971 0.0-0.31 gallic y = 6.2295x + 0.017 0.9997 0.0-0.214 protocatechuic y = 5.8417x + 0.0177 0.9989 0.0-0.15 caffeic y = 7.1463x + 0.0075 0.9995 0.0-0.1 egcg y = 4.5783x + 0.0134 0.9995 0.0-0.2 p-cumaric y = 3.0353x + 0.0227 0.9981 0.0-0.25 baicalin y = 4.0829x + 0.0251 0.9981 0-0.2 o -coumaric y = 3.2019x + 0.0342 0.9975 0.0-0.3 naringenin y = 4.0863x + 0.0314 0.9970 0.0-0.2 vanillic y = 3.2699x + 0.0253 0.9979 0.0-0.2575 naringin y = 0.669x + 0.0516 0.9886 0-1 homovanillic y = 3.6029x + 0.0373 0.9957 0.0-0.22 arbutin y = 1.9775x + 0.0199 0.9989 0.0-0.43 salicilic y = 0.0678x + 0.0436 0.9739 0-5 rutin y = 2.2528x + 0.0316 0.9975 0.0-0.38 syringic y = 2.0896x + 0.0103 0.9996 0.0-0.416 chryzin y = 2.9379x + 0.0313 0.9964 0.0-0.255 ferulic y = 3.1078x + 0.0213 0.9978 0.0-0.25 quercitin y = 6.264x + 0.0044 0.9994 0.0-0.12 isoferulic y = 3.3648x + 0.0441 0.9962 0.0-0.3 catechine y = 4.6179x +0.0127 0.9987 0.0-0.1 tannic y = 4.4556x + 0.0088 0.9994 0.0-0.15 morin y = 3.8719x + 0.019 0.9989 0.0-0.22 homogentisin y = 3.7893x + 0.0073 0.9990 0.0-0.22 coniferyl aldehyde y = 3.4441x + 0.0186 0.9977 0.0-0.22 gentisin y = 6.3517x + 0.0169 0.9993 0.0-0.15 metferulald y = 2.1977x + 0.021 0.9978 0.0-0.4 ellagic y = 4.4621x + 0.0175 0.9988 0.0-0.206 3,5dimetoxy cinald y = 3.4992x + 0.0199 0.9991 0.0-0.25
Converting factors for individual phenolic compounds determining using F-C method sinapic acid gallic acid catechine sinapic acid 1.000 0.418 0.573 gallic 2.358 1.000 1.358 caffeic 2.665 1.132 1.536 p -cumaric 1.183 0.496 0.679 o -cumaric 1.289 0.542 0.740 vanillic 1.281 0.538 0.735 homovanillic 1.450 0.611 0.833 salicilic 0.151 0.054 0.082 syringic 0.783 0.325 0.447 ferulic 1.205 0.506 0.692 isoferulic 1.387 0.584 0.797 tannic 1.663 0.702 0.956 homogentisic 1.408 0.593 0.809 gentisic 2.403 1.019 1.385 ellagic 1.698 0.717 0.977 chlorogenic 1.293 0.543 0.742 egcg 2.215 0.939 1.276 baicalin 1.726 0.729 0.993 naringenin 1.584 0.668 0.911 naringin 1.609 0.679 0.925 arbutin 0.406 0.163 0.229 rutin 0.777 0.322 0.444 chryzin 0.924 0.385 0.529 protocatechuic 1.179 0.495 0.676 quercitin 2.323 0.985 1.338 coniferyl aldehyde 1.738 0.734 1.000 metferulald 1.483 0.625 0.852 morin 1.321 0.555 0.759 3,5dimetoxy cinald 0.863 0.359 0.494 catechine 1.347 0.566 0.773
Chemical structure of DPPH and DPPH - H DPPH + AH DPPH-H + A
Kinetic behaviour of ascorbic acid Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT Food Sci. Technol., 1995, 28, 25-30.
Kinetic behaviour of quaiacol Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT Food Sci. Technol., 1995, 28, 25-30.
Disappearance of DPPH radical as a function of the moles of zingerone/mole DPPH radical Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT Food Sci. Technol., 1995, 28, 25-30.
Classification of antiradical efficiacies and stechiometry, according to kinetic behavior Kinetic behavior Compound ARP Stoichiometric value Number of reduced DPPH Rapid Isoeugenol Ascorbic acid Isoascorbic acid 1.94 3.70 3.70 1.03 0.54 0.54 0.97 1.85 1.85 Intermediate δ-tocopherol Rosmarinic acid 4 6.90 0.50 0.30 2 3.33 Slow Phenol Coumaric acid Vanillin Vanillic acid Γ-resorcylic acid Ferulic acid Eugenol Zingerone Guaiacol BHA BHT Protocatechuic acid Caffeic acid Gentisic acid Gallic acid 0.002 0.02 0.05 0.17 0.36 2.33 3.7 3.7 4 4.17 4.20 7.14 9.1 11.1 12.5 270 98 44 11.8 5.6 0.86 0.54 0.54 0.5 0.38 0.38 0.28 0.22 0.18 0.16 <1 <1 <1 <1 <1 1.16 1.85 1.85 2 2.63 2.63 3.6 4.54 5.6 6.25 Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT Food Sci. Technol., 1995, 28, 25-30.
Antiradical activity of selected phenolic acids against DPPH radical Phenolic acid EC 50 (µmol/assay) ARP Relative ARP (%) Gallic Gentisic Syringic Caffeic Protocatechuic Sinapic Ferulic Isoferulic Vanillic p-coumaric o-coumaric m-coumaric Salicylic p-hydroxybenzoic 0.0237 0.0292 0.0427 0.0478 0.0574 0.0724 0.0927 5.68 14.37 66.29 130.05 > 300 > 800 > 800 42.19 34.25 23.42 20.92 17.42 13.81 10.79 0.176 0.069 0.015 0.008 <0.003 <0.001 <0.001 100 81.81 55.51 49.58 41.31 32.73 25.57 0.42 0.16 0.04 0.02 <0.007 <0.002 <0.002 Karamać M., Kosińska A., Pegg R.B.: Comparison of radical-scavenging activities for selected phenolic acids. Pol. J. Food Nutr. Sci., 2005, 55, 165-170.
Stoichiometry n = 1 DPPH + AH DPPH 2 + A A + A A-A -------------------------------------------------------- 2 DPPH + 2AH 2 DPPH 2 + A-A
Stoichiometry n = 2 DPPH + AH DPPH 2 + A DPPH + A DPPH-A ----------------------------------------------------- 2 DPPH + AH DPPH 2 + DPPH-A
Stoichiometry n = 3 DPPH + AH DPPH 2 + A DPPH + A DPPH-A DPPH + DPPH-A DPPH 2 -Q -------------------------------------------------------------- 3 DPPH + AH DPPH 2 + DPPH-Q
Stoichiometry n = 4 DPPH + AH DPPH 2 + A DPPH + A DPPH-A (P 1 ) DPPH + P 1 DPPH 2 + P 2 DPPH + P 2 DPPH 2 + P 3 ---------------------------------------------- 4 DPPH + AH 3 DPPH 2 + P3 Vessela D. Kancheva: Natural antioxidants for the preservation of oils kinetic studies and mechanisms. Eur. J. Lipid Sci. Technol. in press.
Antiradical activity against DPPH radical 1.4 1.4 1.2 1.2 Absorbance at 517 nm 1 0.8 0.6 0.4 Absorbance at 517 nm 1 0.8 0.6 0.4 0.2 Extract Fraction II 0 0 0.004 0.008 0.012 0.016 0.02 0.2 Fraction I 0 0 0.02 0.04 0.06 0.08 0.1 Content (mg/assay) Content (mg/assay)